Functional distribution of epithelial sodium channels and water channels in the amphibian skin

为了定位上皮性钠通道，我们从A6肾细胞系中克隆了一种针对上皮钠通道α亚基胞外环肽的多克隆抗体(αxENaC)。在荒漠蟾蜍中，αENaC成功地定位于颗粒细胞层最浅层细胞的顶端细胞膜。接下来，我们使用了一种抗体来对抗从树蛙腹面皮肤中克隆的水通道蛋白(水通道蛋白-2：AQP-H2)的C端肽。该抗体识别30.0kD的蛋白，免疫染色主要在斑点巴氏杆菌的腹部盆腔皮肤。在皮肤的其他区域，如胸部和背部皮肤，没有观察到免疫反应，这与利用骨盆皮肤的沙漠蟾蜍的吸水行为一致。免疫反应在颗粒层上层较强，定位于主细胞顶端细胞膜。腹侧皮肤暴露于抗利尿激素(AVT，10^&lt；-8&gt；M)促进了顶膜更强烈的免疫反应。结果表明，沙漠蟾蜍能在吸水底物中检测到钠离子。

To localize the epithelial sodium channel (ENaC) we raised a polyclonal antibody against a peptide in the extracellular loop of the α subunit of Xenopus ENaC (αxENaC) that had been cloned from A6 kidney cell line. In desert toads, Bufo alvarius, the α ENaC was successfully localized in the apical cell membrane of the most superficial cells in the granulosum cell layer. Next, we used an antibody against, the C-terminal peptide of a water channel protein (aquaporin-2 : AQP-h2) that had been cloned from the ventral skin of the tree frog, Hyla japonica. The antibody recognized a 30.0 kD-protein and immunostained predominantly in the abdominal pelvic skin of B.alvarius. No immunoreactivity was seen in the other area of the skin such as the pectoral and dorsal skins, which is consistent to a water absorption behavior of the desert toads that utilize the pelvic skin. The immunoreactivity was intense in the upper layer of the stratum granulosum and was localized to the apical cell membrane of the principal cells. Exposure of the ventral skin to anti-diuretic hormone (AVT, 10^<-8>M) facilitated more intense immunoreactivity in the apical membrane. Results explain that the desert toad can detect sodium ions in the substrate of water absorption.